The genus Agrobacterium (for a recent review see Gelvin 2003) has been divided into a number of species. However, this division has reflected, for the most part, disease symptomology and host range. A. radiobacter is an “avirulent” species, A. tumefaciens causes crown gall disease, A. rhizogenes causes hairy root disease, A. rubi causes cane gall disease, and A. vitis causes galls on grape and a few other plant species (Otten 1984; Smith and Townsend 1907; Hildebrand 1934; for review on A. rhizogenes see Nilsson and Olsson, 1997). Although Bergey's Manual of Systematic Bacteriology still reflects this nomenclature, classification is complex and confusing. The disease symptomology is largely due to the transfer, integration, and expression in the plant cell genome of DNA (T-DNA) originating from large plasmids called Ti (tumor inducing) and Ri (root inducing) plasmids (Van Laerebeke 1974; Chilton 1977, 1982; Moore 1979; White 1982; Tepfer 1983; Nester 1984). Curing a particular plasmid and replacing this plasmid with another type of tumorigenic plasmid can alter disease symptoms. For example, infection of plants with A. tumefaciens C58, containing the nopaline-type Ti plasmid pTiC58, results in the formation of crown gall teratomas. When this plasmid is cured, the strain becomes nonpathogenic. Introduction of Ri plasmids into the cured strain “converts” the bacterium into a rhizogenic strain (Lam 1984, White 1980). Furthermore, one can introduce a Ti (tumor-inducing) plasmid from A. tumefaciens into A. rhizogenes; the resulting strain incites tumors of altered morphology on Kalanchoe plants (Costantino 1980). Thus, because A. tumefaciens can be “converted” into A. rhizogenes simply by substituting one type of oncogenic plasmid for another, the term “species” becomes meaningless. Thus, in recent years the method to distinguish the bacteria strains by their crown gall or hairy root phenotype does not seem to be appropriate anymore, since these features are only linked to the extra-chromosomal plasmid. Genomic DNA analysis revealed that some strains formerly classified as A. rhizogenes are more related to A. tumefaciens and vice versa.
A more meaningful classification system divides the genus Agrobacterium into “biovars” based on growth and metabolic characteristics (Keane 1970). Using this system, most A. tumefaciens and A. rubi (Tighe 2000) strains belong to biovar I, A. rhizogenes strains fit into biovar II, and biovar III is represented by A. vitis strains. More recently, yet another taxonomic classification system for the genus Agrobacterium has been proposed (Young 2001). The recent completion of the DNA sequence of the entire A. tumefaciens C58 genome (which is composed of a linear and a circular chromosome, a Ti plasmid, and another large plasmid (Goodner 1999, 2001; Wirawan 1996) may provide a starting point for reclassification of Agrobacterium “strains” into true “species.” A recent classification based on RAPD (random amplified polymorphic DNA) reflects the genomic differences and is providing a “family” tree for several Agrobacterium strains (Llop 2003). A modified classification scheme was proposed by Sawada (Sawada 1993).
Although the genetic background of Agrobacteria is little explored, extensive knowledge already exists about the functionality of their Ti or Ri-plasmids in plant infection. Mobilization of the T-DNA requires that the products of genes located elsewhere on the Ti or Ri plasmid, called collectively the vir genes, which are activated by certain elicitors from the wounded plant cells in trans to synthesize and transfer a single-stranded copy of the T-DNA (the T-strand) to the plant cell (Zambryski 1992; Zupan 1995). The T-DNA sequence on the Ti plasmid is flanked by short 24-bp direct repeats (Yadav 1982), which are required for the recognition of the T-DNA (Wang 1984). Sequences immediately surrounding these borders appear to be involved in the polarity of T-strand synthesis, which initiates at the right border (Wang 1987). Foreign DNA flanked by T-DNA border sequences can be transferred into plant cells using A. tumefaciens as the vector (Hernalsteens 1980). Inactivation or removal of the native T-DNA genes involved in hormone synthesis would render the A. tumefaciens incapable of producing the crown gall disease symptoms. This process of inactivating or removing genes responsible for disease symptoms is termed “disarming.” The first methods of A. tumefaciens engineering involved the simultaneous disarming and introduction of the desired gene, since the introduced gene directly replaced the genes in the T-DNA By a method termed “homogenotization” (Matzke and Chilton, 1981), the native T-DNA of the Ti plasmid was replaced with a desired gene for transformation. Another strategy developed for engineering A. tumefaciens involved cloning the desired gene into a cointegrative intermediate vector, which contained a single region of T-DNA homology and a single border sequence. In this system, the sequences are recombined by a single-crossover event (Horsch 1985), which results in the entire vector, including the gene of interest, being integrated. Cointegrative systems pair in regions of homology between the T-DNA region of the Ti plasmid and the DNA sequence on the introduced integrative vector. One example of a useful cointegrative plasmid is pGV3850, a Ti plasmid from a nopaline strain (C58), from which the entire T-DNA region between the borders was replaced with pBR322, thus offering a recombination site for any gene construct containing pBR322 homology (Zambryski 1983).
Upon the discovery that T-DNA does not have to be on the same plasmid as the vir genes (de Framond 1983; Hoekema 1983, 1985), the binary vector was developed. A binary vector is maintained in the A. tumefaciens separate from the Ti plasmid, and contains the gene of interest and a plant selectable marker gene between T-DNA border sequences. These vectors offer a great degree of flexibility, since they do not require a specifically engineered Ti plasmid with a homologous recombination site. For that reason, any disarmed A. tumefaciens strain can be used to transfer genes for any binary vector. Owing to their versatility, binary vectors are currently the preferred intermediate vectors for cloning genes destined for Agrobacterium-mediated transformation in plants. However, any A. tumefaciens strain to be used with binary vectors must have its own Ti plasmid disarmed, especially if the target plant species is inefficiently transformed via A. tumefaciens. Otherwise, the desired gene from the binary vector will be co-transformed with the oncogenic phytohormone genes from the native T-DNA of the bacteria, thereby reducing transformation efficiency of the desired gene and also producing the tumorigenic disease symptoms in many of the target cells and thereby preventing the differentiation of these cells into normal plants.
Disarming wild-type A. tumefaciens strains for general use with binary vectors has involved, in some cases, a form of homogenotization. An intermediate construct containing a marker gene flanked by Ti plasmid sequences that are homologous to regions that lie outside the T-DNA, is introduced into the wild-type A. tumefaciens by bacterial conjugation (Hood 1986, 1993). Whereas disarmed A. tumefaciens strains typically have their entire T-DNA sequences removed, it has also been demonstrated that T-DNA mobilization can be inactivated by removal of the right border sequence: reports from work with nopaline-type strains of A. tumefaciens show that the right border of T-DNA is necessary for gene transfer, whereas the left border is not (Joos 1983; Peralto and Ream 985; Shaw 1984; Wang 1984). Agrobacterium tumefaciens has a diverse dicot host range, and additionally some monocot families (De Cleene 1976; Smith 1995). There are several different strains of A. tumefaciens, each classified into octopine-type, nopaline-type, and L,L-succinamopine-type, named after type of opine synthesized in the plant cells they infect. These strains have comparable, although not identical, host ranges and disarmed versions of many types of A. tumefaciens have been used successfully for gene transfer into a variety of plant species (van Wordragen 1992; Hood 1993).
Agrobacterium rhizogenes strains are classified the same way A. tumefaciens strains are. Typically, they are classified by the opine they produce. The most common strains are agropine-type strains (e.g., characterized by the Ri-plasmid pRi A4), mannopine-type strains (e.g., characterized by the Ri-plasmid pRi8196) and cucumopine-type strains (e.g., characterized by the Ri-plasmid pRi2659). Some other strains are of the mikimopine-type (e.g., characterized by the Ri-plasmid pRi1724). Mikimopine and cucumopine are stereo isomers but no homology was found between them on the nucleotide level (Suzuki 2001).
Soybean (Glycine max L. Merr.) has proven to be very difficult to transform with A. tumefaciens, at least in part because it is refractory to infection by wild-type A. tumefaciens. Comparative studies with a number of soybean cultivars and A. tumefaciens strains suggest that soybean susceptibility to A. tumefaciens is limited, and is both cultivar- and bacterial strain dependent (Bush 1991; Byrne 1987; Hood 1987). The problems with soybean recalcitrance to A. tumefaciens are further complicated by the difficulty of working with soybean in tissue culture. Despite some advances to date, however, Agrobacterium-mediated transformation in soybean remains inefficient and labor-intensive, and methods for improving that efficiency are continually being sought.
As mentioned earlier, some A. tumefaciens strains infect soybean more readily than others. One strain, A281, is a supervirulent, broad host-range, L,L-succinamopine-type A. tumefaciens with a nopaline-type C58 chromosomal background, containing the L,L-succinamopine-type Ti plasmid, pTiBo542 (Hood 1987). Disarming this strain has produced EHA101 and EHA105, strains now widely used in conjunction with soybean transformation (Hood 1986, 1987). Various other disarmed Agrobacterium strains are described (A208, U.S. Pat. No. 5,416,011; LBA 4404, WO 94/02620). Hood et al., (1993) disclose the disarming of three Ti plasmids: one each of the octopine, nopaline and L,L-succinamopine types. Agrobacterium tumefaciens strains A281 and EHA101 are disclosed as able to transform soybean. The disarming derivative of plasmid pTiBo542 from strain A281 is disclosed and designated pEHA105.
Agrobacterium rhizogenes Ri-transformed plants of several plant species have a characteristic phenotype, with shortened internodes, wrinkled leaves, and an abundant root mass with extensive lateral branching (Tepfer 1984). The rol genes in Ri T-DNA induce changes in sensitivity to plant hormones and/or in the metabolism of plant hormones (Maurel 1994; Moritz and Schmülling 1998; Nilsson 1997; Shen 1988). Furthermore, transformation of plant tissues by infection with A. rhizogenes increases the production of certain metabolites (Ermayanti 1994; Mano 1986; Sim 1994).
Native, “armed” Agrobacterium rhizogenes K599 (pRi2659) is capable to induce hairy root formation in a variety of soybean cultivars including Jack, Williams 82, Cartter, Fayette, Hartwig, Mandarin, Lee 68, Peking, and PI437654 (Cho 2000).
In the case of A. rhizogenes, the mannopine Ri plasmid of strain 8196 possesses a single T-region which does not share homology with any of the pTi T-DNA oncogenes (Lahners 1984). This observation suggests that a novel mechanism, different from that due to tms expression in tmr Ti mutants, is responsible for root induction by this strain. In the case of the agropine strains such as A4, two distinct regions of the Ri plasmid are transferred to the plant genome: the TL-region and the TR-region (Huffman 1984; Jouanin 1984; White 1985). The size of the TL-DNA encountered in plants transformed by strain A4 is quite constant, while the length of the TR-DNA is more variable. Hybridizations with the T-regions of A. tumefaciens revealed homology in the pRi TR-region with genes of the TR-DNA of octopine Ti plasmids that are involved in agropine synthesis. Amongst the common pTi oncogenes, homology was found only with the fm loci (Willmitzer 1982; Huffman 1984; Jouanin 1984), suggesting a possible role for TR-DNA directed auxin synthesis in root induction, even if the tms-like genes are not found in the genome of all regenerated transformed plants (Taylor 1985; Jouanin 1986a). The TL region, in contrast, does not hybridize with genes of pTi T-DNA (Jouanin 1984). The TL-DNA sequence established by Slightom et al. (1986) confirms this absence of homology at the nucleotide level. However, the TL-DNA is highly homologous to the single T-region of the mannopine pRi8196 and might therefore be capable of inducing transformed roots.
Vilaine et al. (Vilaine 1987) have demonstrated that the transfer of TL-DNA alone, as well as the transfer of TR-DNA alone, does lead to root induction on infected plant fragments, suggesting the existence of two independent molecular mechanisms for root induction on agropine type Ri plasmids. Vilaine et al. are further describing disarming the agropine-type Agrobacterium rhizogenes A4RS strain by deleting the TL, TR, or both the TL and TR regions from the Ri-plasmid pRiA4 resulting in A. rhizogenes strain RS (pRiB278b). Described is conjugation of the disarmed Ri plasmids with cosmids carrying the TL or TR region thereby “rescuing” the hairy root phenotype. No use for gene transfer of said disarmed A. rhizogenes strain is disclosed.
While Agrobacterium tumefaciens mediated plant transformation has become a standard in the plant biotech industry for many plants species, use of Agrobacterium rhizogenes is only rarely made. To date, only native “armed” Agrobacterium rhizogenes strains were employed to incorporate foreign genes into plants (e.g., Narayanan 1999; Kouchi 1999). Since A. rhizogenes can also transfer the T-DNA of binary vectors ‘in trans’, the Ri plasmid has been used as a vector for the introduction of foreign DNA into dicotyledonous plant species (Bevan 1984; Simpson 1986; Hamill 1991). However, the Agrobacterium rhizogenes strains employed in these disclosures are “armed” (by comprising their native Ri plasmids) and are still able to cause the hairy root phenotype (see e.g., Narayanan 1999).
Although some of the problems linked to the plant transformation have been overcome by the methods described in the art, there is still a significant need for improvement and alternative procedure. Although significant advances have been made in the field of Agrobacterium-mediated transformation methods, a need continues to exist for improved methods to facilitate the ease, speed and efficiency of such methods, especially also for transformation of monocotyledonous plants and dicotyledonous plants which are recalcitrance to transformation with standard A. tumefaciens strains. Therefore, it was the objective of the present invention to provide an alternative method which offers an improved transformation efficiency for a broad variety of plant species. This objective is solved by the present invention.